Ontogeny of macrophage subpopulations and Ia-positive dendritic cells in pulmonary tissue of the rat

Summary The development of macrophage subpopulations and dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gutassociated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.Fellow of the Royal Netherlands Academy of Arts and Sciences     

Immunohistological localization of alpha 1-microglobulin in normal rat tissues

The tissue distribution of rat alpha 1-microglobulin (alpha 1-m) was studied by indirect immunofluorescence in various rat tissues using a polyvalent rabbit antiserum to the purified antigen and a monoclonal antibody (H23) to the human homologue, in parallel with a polyclonal anti-rat IgA antiserum. It was found that all tissues stained by anti-IgA were also alpha 1-m positive; these tissues included tissues of the stomach, duodenum, ileum, colon, pancreas, trachea, esophagus and jejunum. However, the observation that IgA plasma cells as well as secretory cells, while positively stained by anti-IgA, are alpha 1-m negative suggests that the association between IgA and alpha 1-m occurs at a postsecretory stage, after the IgA molecules have been transported across the epithelial cells. Additionally, hepatocytes were intensely stained by anti-alpha 1-m antibodies, indicating that the liver, as already suggested by metabolic studies on isolated guinea-pig liver explants, may be responsible for the synthesis of this protein. Among lymphoid tissues, an intense and homogeneous staining was observed in the thymus and the white pulp of the spleen. Sections of lymph nodes, however, showed differential staining; apart from a few isolated dendritic cells in the mantle region of the lymphoid follicles, the germinal centers and medullary cords showed no staining with anti-alpha 1-m antibodies. The paracortical cells, macrophages in the subcapsular sinus, and interfollicular lymphocytes showed intense cytoplasmic staining with anti-alpha 1-m antibodies. In other tissues, macrophages, monocytes, tissue histiocytes, and dendritic cells were alpha 1-m positive. Although they confirm the presence of alpha 1-m in the lymphoid tissues, as already reported in man, these results show that the protein is also present in hepatocytes and in exocrine fluids containing IgA. Since alpha 1-m, like secretory component, can bind to IgA to form stable complexes, these two heavily glycosylated proteins may have similar biologic properties.     

A novel monoclonal antibody, RM-4, specifically recognizes rat macrophages and dendritic cells in formalin-fixed, paraffin- embedded tissues

Summary An anti-rat macrophage/dendritic cell monoclonal antibody, RM-4, was produced using a homogenate of silica-induced lung granulomas of rat as immunogen. Immunohistochemistry demonstrated that RM-4 was specific for macrophage and dendritic cell populations residing in various organs and tissues. It did not react with any cells other than macrophage/dendritic cells. In the double staining of the spleen, RM-4-positive macrophages showed wider distribution than those of the four other anti-rat macrophage monoclonal antibodies compared. The immunoreactivity of RM-4 was well preserved not only in frozen sections but also in formalin-fixed, paraffin-embedded tissues. The isotype of the monoclonal antibody was IgG₁ kappa and its antigen molecular weight was 46 kDa. Immunoelectron microscopy revealed positive reaction products for RM-4 on the membrane of endosomes and lysosomes in macrophages and epidermal Langerhans cells. Reaction intensity increased after thioglycolate elicitation or endocytosis regardless of ingested materials. From these data, it is concluded that RM-4 recognizes a membrane protein of endolysomes in macrophages and dendritic cells. The antigen may play a role in endolysosomal processing. RM-4 is considered to be a useful tool not only for identifying macrophage/dendritic cells both in frozen and paraffin-embedded tissues, but also for evaluating their endolysosomal processing. This revised version was published online in November 2006 with corrections to the Cover Date.     

Identification of tissue histiocytes on paraffin sections by a new monoclonal antibody

A mouse monoclonal antibody (MAC 387) with specificity for monocytes and tissue histiocytes was produced by immunization of a BALB/c mouse with peripheral blood monocyte components derived by affinity chromatography of detergent-solubilized monocyte material on Sepharose 4B coupled to rabbit anti-monocyte antibodies. MAC 387 strongly stained the cytoplasm of cells of the monocyte/macrophage series on paraffin sections after controlled trypsinization of sections. The antibody showed broad reactivity for a variety of tissue histiocytes, including infiltrating and reactive histiocytes, alveolar macrophages, Kupffer cells, follicle-center macrophages, splenic red pulp macrophages, tumor-infiltrating macrophages, sinus histiocytes, epithelioid giant cells (variably), and cases of histiocytosis X and dermatopathic lymphadenopathy. Molecular weight data obtained by Western blotting, immunoprecipitation, and immunoaffinity-purification revealed that the antigen was present in different forms in the monocyte and granulocyte. In the granulocyte, free alpha (Mr 12 KD) and beta (Mr 14 KD) chains expressing the MAC 387 epitope were found together with associations of one alpha and one beta chain linked by disulfide bonds to yield a heterodimer of Mr 26 KD. In the monocyte, free alpha and beta chains are not found, but instead the heterodimer and associations of two (Mr 56 KD) and four (Mr 112 KD) heterodimers are disulfide-linked together. This new monoclonal reagent should have particular value for identification of tissue histiocytes in routine paraffin sections and particularly for demonstration of histiocytes in malignant lymphomas.     

Improved Immunohistochemical Visualization of Helper/Inducer T-Cells by the Simultaneous Application of two Noncrossblocking Monoclonal Antibodies

We have studied the intensity of staining of helper/inducer T-cells in lymph node and tonsillar tissue using two commercially available monoclonal antibodies (OKT4 and Leu3a) with the indirect immunoperoxidase method. Paracortical and mantle zone helper/inducer T-cells were easily visualized by both monoclonal antibodies, but T-cells in the follicular center, though stained by Leu3a, were hardly demonstrable by OKT4. Excellent staining was obtained in the indirect immunoperoxidase procedure by incubating the sections with a 1:1 mixture of the two monoclonal antibodies which gave bright staining of individual cells throughout the lymphoid tissue. Dilution of the primary antibodies by 1:200 did not affect the results. It is concluded that the simultaneous application of OKT4 and Leu3a as primary antibodies in the indirect immunoperoxidase procedure is the method of choice for the in situ demonstration of helper/inducer T-cells.     

Differential immunohistochemical resolution of the human mononuclear phagocyte system

Four new monoclonal antibodies, termed Ki-M1, Ki-M2, Ki-M3, and Ki-M4, were developed for distinguishing macrophage subgroups. Purified lysosomes of cells of stimulated U-937 cell line were used as immunogen. Specificity control was performed by staining unfixed cryostat sections of fresh human tissue with an immunohistochemical method, which allowed reliable recognition of reactive structures. Ki-M1 reacted with macrophages of lymphoid tissue, lung, and serous cavities. Ki-M2 recognized Kupffer cells and splenic macrophages. Both monoclonal antibodies reacted with interdigitating reticulum cells and Langerhans cells, which are thought to be accessory cells of the T-cell immune response. Accessory cells of the B-cell immune response, on the other hand, showed reactivity with Ki-M3 and Ki-M4. Thus, analogous to T lymphocytes, the human mononuclear phagocyte system (MPS) can be subdivided into different subgroups with the aid of appropriate monoclonal antibodies.     

Improved immunohistochemical visualization of helper/inducer T-cells by the simultaneous application of two noncrossblocking monoclonal antibodies

We have studied the intensity of staining of helper/inducer T-cells in lymph node and tonsillar tissue using two commercially available monoclonal antibodies (OKT4 and Leu3a) with the indirect immunoperoxidase method. Paracortical and mantle zone helper/inducer T-cells were easily visualized by both monoclonal antibodies, but T-cells in the follicular center, though stained by Leu3a, were hardly demonstrable by OKT4. Excellent staining was obtained in the indirect immunoperoxidase procedure by incubating the sections with a 1:1 mixture of the two monoclonal antibodies which gave bright staining of individual cells throughout the lymphoid tissue. Dilution of the primary antibodies by 1:200 did not affect the results. It is concluded that the simultaneous application of OKT4 and Leu3a as primary antibodies in the indirect immunoperoxidase procedure is the method of choice for the in situ demonstration of helper/inducer T-cells.     

Characterization of rapidly adhering amniotic fluid cells by combined immunofluorescence and phagocytosis assays.

Culture of human amniotic-fluid cells from cases of fetal neural tube defects produces a population of rapidly adhering cells that were initially thought to be macrophages and later interpreted to be of neural origin. In this study double and triple labeling systems for the simultaneous detection of glial and macrophage differentiation marker antigens have been used to demonstrate that rapidly adhering cells cannot be considered a homogeneous population but instead represent two distinct cell types. One of these cell populations is of glial origin and shows specific staining for glial fibrillary acidic protein, while the other population is monocyte-derived macrophages which express marker antigens recognized by Leu M3, KiM7, and Dako antimacrophage monoclonal antibodies.     

Antibodies to phospholipids and liposomes: binding of antibodies to cells

Binding of two monoclonal anti-liposome antibodies to the surface of cultured murine peritoneal macrophages was investigated by indirect immunofluorescence and enzyme-linked immunosorbent assay. Neither antibody bound to cultures of freshly explanted, nonadherent macrophages, but immunoreactivity was observed following cell adherence to tissue culture plastic. Fluorescent microscopic evaluation revealed heterogeneity in staining patterns of the antibodies on adherent cells. Binding both to viable and fixed adherent macrophages was observed even after a 10,000-fold dilution of antibody. Treatment of adherent macrophage cultures with trypsin increased antibody binding. Further treatment of trypsinized-macrophages with alkaline phosphatase or neuraminidase did not affect antibody binding, but phospholipase D and, to a greater extent, phospholipase C resulted in a marked decrease in cellular binding. The data indicate that antibodies produced against liposomes appear to bind to surface phospholipids of macrophages, but binding can be influenced by the physiological state of the macrophage and overlying cell surface proteins.     

Distribution of Leu 7 (HNK-1) antigen in human digestive organs: an immunohistochemical study with monoclonal antibody

Summary Leu 7 immunoreactivity was demonstrated with the indirect peroxidase-labelled antibody method on frozen and paraffin-embedded tissue sections of human digestive organs. Anti-Leu 7 monoclonal antibody, which allegedly detects mononuclear cells with natural killer or killer activity, recognized lymphoid cells among intestinal epithelial cells and in the germinal centres of solitary lymphoid follicles of small and large intestine, and a few in gallbladder, liver and the lamina propria of the intestine. In addition, peripheral nerve fibres, endocrine cells in the gut and pancreas and carcinoid and islet cell tumours were also positively stained. At the ultrastructural level, Leu 7 antigen was localized on the plasma membrane of granulated lymphoid cells in the gut mucosa and on the secretory granules of intestinal endocrine cells. In normal pancreas, Leu 7 immunoreactivity was demonstrated in most cells containing pancreatic polypeptide and in many cells containing somatostatin or glicentin. Insulin-containing cells, however, lacked Leu 7 immunoreactant. These findings were obtained in both frozen sections and paraffin-embedded sections. The possible cross-reactivities of monoclonal antibodies are discussed as they raise an important caveat in immunohistochemical studies using these antibodies.     

Immunophenotypic characterization of primary and secondary lymphoid follicles

The need for an immunophenotypical referential framework relative to lymphoid follicle has led us to apply a panel of monoclonal and polyclonal antibodies, by means of a sensitive immunostaining method. Lymphoid follicle is an immunophenotypically complex structure made up of three lymphoid populations (B, being its bulk, and a few T and NK cells), dendritic reticulum cells (DRCs) and Flemming's macrophages. Follicular B population is To 15 +, B1+, OKB 7 +, HLA-DR + and C3bR +. In secondary follicles there are differential characteristic reactivities for each topographic compartment: Mantle zone is positive for OKB 2 and surface IgM (sIgM) and IgD (sIgD); germinal center (GC) clear zone (with centrocytic predominance) for OKT 9, sIgM and weakly for OKB 2; and GC dark zone (with centroblastic predominance) only for OKT 9. In sections, OKT 10 allows one to see immunoblasts and plasma cells, the latter being with lymphoplasmacytoid cells the only intracytoplasmic immunoglobulin holders. 10% of GC lymphocytes are T cells, almost exclusively T-helper (Leu 3a +). Another 10% to 15% of lymphoid cells are Leu 7 (HNK-1) +. In histological sections, DRCs are specifically marked with R4/23 and Flemming's macrophages with anti-alpha1-antitrypsin and anti-alpha1-antichymotrypsin antibodies, both populations being negative to OKM 1 and OKM 5.     

Defining the molecular target of an antibody derived from nuclear extract of Jurkat cells using protein arrays

Many diagnostic antibodies are generated by immunization with whole cells or cell extracts and are shown by screening on tissue sections to label specific cell populations. However, their target molecule then needs to be identified, and this can be technically demanding. Here we describe the use of protein arrays to define the targets of new or uncharacterized monoclonal antibodies. The technique involves screening protein arrays containing thousands of recombinant human proteins. An initial experiment showed that a well-characterized monoclonal antibody against nucleophosmin identified 22 clones on the array encoding this protein. Next, the antibody JJ166, for which the antigen had not yet been identified, was screened. This antibody was generated by immunizing with a nuclear extract of Jurkat cells and was detected in immunohistochemistry due to its distinctive nuclear staining of lymphoid tissue cells. However, its molecular target had remained unidentified using traditional approaches. A protein array screen rapidly identified the mitotic spindle-associated molecule NUMA1 (nuclear mitotic apparatus protein 1). To confirm this putative specificity, JJ166 was shown to react with COS-1 cells transiently transfected with the complementary DNA for NUMA1. Furthermore, a commercially available antibody against NUMA1 showed nearly identical staining in immunohistochemistry on human tissue and cells. Overall, this method represents an effective and quick strategy for defining the protein targets of new or uncharacterized monoclonal antibodies identified as having diagnostic or other potential value on the basis of their immunostaining patterns.     

A monoclonal antibody to a novel differentiation antigen on human macrophages associated with the down-regulatory phase of the inflammatory process

A monoclonal antibody (RM3/1), raised by immunizing mice with human monocytes, is described which detects a surface antigen on about 20% of freshly isolated peripheral blood monocytes and is increasingly expressed upon cultivation, reaching a maximum between day 2 and 3. By incubation of monocytes with interferon-gamma, 12-O-tetradecanoylphorbol-13-acetate and lipopolysaccharide, antigen expression is decreased but strongly enhanced after incubation with dexamethasone. In cryostat sections of normal tissue, the antibody detects histiocytes in the skin, Kupffer cells in the liver, few alveolar macrophages in the lung, macrophages in the red pulp of the spleen and in the cortex of the thymus, and many macrophages in the placenta. In acute inflammatory tissue, e.g. gingivitis, the antigen is preferentially expressed by macrophages appearing late in the inflammatory process. In chronic inflammation, e.g. BCG granulomas and rheumatoid arthritis, RM3/1-positive macrophages are seen to varying degrees. Double-staining experiments with the antigen 25F9, specific for resting mature macrophages, revealed that RM3/1 and 25F9 are expressed by distinct populations in normal and acute inflammatory tissues. From this it is concluded that the antibody RM3/1 specifically detects a macrophage phenotype which seems to be associated with the healing phase of the inflammatory process.     

Heterogeneity of rat macrophages recognized by monoclonal antibodies: an immunohistochemical and immunoelectron microscopic study

Three monoclonal antibodies, designated RM-1, TRPM-1, and TRPM-2, were raised against rat peritoneal macrophages. By the immunoperoxidase method, antigens recognized by these antibodies were distributed throughout most tissue and free macrophages examined, including those of splenic red pulp, lymphatic sinus, connective tissue, and peritoneal cavity, as well as Kupffer cells of liver and alveolar macrophages. The numbers of positive cells were different for each antibody. RM-1 and TRPM-1 were also reactive with interdigitating cells (IDCs) in the thymus-dependent area and with Langerhans cells in the skin, whereas TRPM-2 failed to demonstrate IDCs in thymic medulla and Langerhans cells. The reactions of each antibody were observed by immunoelectron microscopy in the different ultrastructural compartments of the cells. RM-1 recognized a cell surface antigen; reaction products for TRPM-1 were found on a part of the cell membrane and in the cytoplasmic vacuoles; and those of TRPM-2 were present along the nuclear envelope and intracytoplasmic vacuoles. These antibodies seem to be useful not only for the detection of macrophages in tissue sections but also for investigation of macrophage heterogeneity in different tissues.     

Reticulum cells in the ontogeny of nasal-associated lymphoid tissue (NALT) in the rat

This study concerns the ontogeny of reticulum cells (RC) in the nasal-associated lymphoid tissue (NALT) of Wistar and Brown-Norway rats. A panel of monoclonal antibodies (mAb) directed against RC in peripheral lymphoid organs (antibodies ED10ED15) was used, together with a recently developed antibody ED17, which recognizes macrophages and Langerhans cells. Early in embryogenesis, staining with common connective tissue markers, ED14 and ED15, was found. ED17-positive cells were present before cells positive to ED1, a pan-macrophage marker, or Ia glycoproteins were observed. The first differentiation of reticulum was seen at the day of birth, when ED10 recognized a distinct area in the nasal mucosa. The first T-lymphocytes were found at the same time. Two days after birth, B-cells and ED11-positive cells were present in the NALT area. Fourteen days after birth, T- and B-cell compartments were recognizable. ED10 was found predominantly in the T-cell area and ED11 was mainly confined to the B-cell compartment. We conclude that the development of the NALT is closely accompanied by the phenotypic specialization of the reticulum. This suggests that the reticulum plays an important role in the compartmentalization of NALT tissue and in the retention of lymphocyte subsets within these compartments.     

Production and characterization of monoclonal antibodies to rabbit lymphocyte subpopulations. I. Tissue immunofluorescence and flow cytometric analysis

Nine monoclonal antibodies to rabbit T cells and B subpopulations have been generated from three separate fusions of spleen cells from mice immunized with fractionated populations of rabbit lymphocytes. These monoclonal antibodies, as well as a previously described rabbit T cell monoclonal antibody, 9AE10, have been analyzed by immunofluorescence staining on frozen tissue sections of rabbit thymus, spleen, and appendix. This screening method permits rapid identification of the lymphocyte subdomains in each tissue which is not possible by other screening methods. Each monoclonal antibody selected has a unique tissue staining pattern. Flow cytometric analysis of these monoclonal antibodies, using indirect immunofluorescence techniques on thymocytes, splenocytes, and PBL, revealed varying percentages of positive cells and individual mean fluorescence intensities indicating different epitope densities for each antigen. These monoclonal antibodies are now being used to characterize normal lymphocyte function and the role of specific lymphocyte subpopulations in experimental disease models in the rabbit.     

Expression of class II antigens by subsets of activated T cells

Gene products coded for within the HLA complex play an important role in the control of immune responses. Class I antigens, coded for by the HLA-A, B, and C loci, are expressed by virtually all mononuclear blood cells. Class II antigens, coded for by the DR, DQ, and DP loci, have a more limited tissue distribution. They are expressed by B cells, monocytes, and by activated, but not by resting, T cells. The class II molecules of B cells and antigen-presenting cells have long been of interest to immunologists, since they are involved in the presentation of antigen, in communication between T cells and B cells and between T cells and adherent cells, and in susceptibility to certain diseases. The class II antigens expressed by activated T cells, however, remain largely uncharacterized in terms of their specificity, functional significance, and molecular nature. We have studied the expression of DR and DQ antigens by activated T cells and then examined the expression of DR versus DQ antigens by Leu 2a and Leu 3a subsets of mitogen-activated populations. Our results demonstrated that, as for class II-positive macrophages, the intensity of staining with monoclonal antibodies directed against DR antigens was much greater than that obtained with those directed against DQ antigens. Interestingly, the percentages of Leu 2a- and Leu 3a-positive cells which expressed DR antigens were quite similar, as were the percentages of Leu 2a and Leu 3a cells which expressed DQ. Thus, there does not seem to be preferential expression of DR versus DQ antigens by mitogen-activated T-cell subsets. Finally, though both DR-positive-DQ-positive and DR-positive-DQ-negative populations were detected, few or no DR-negative-DQ-positive cells were observed in these populations.     

Macrophages in the interstitial tissue of the rat testis

Summary Macrophages were identified in the intertubular tissue of the rat testis by loading animals with a particulate vital dye (trypan blue or India ink) and by localizing immunocytochemically a macrophage membrane antigen (MRC W3/25). Leydig cells were identified by the histochemical staining reaction for 3-hydroxysteroid dehydrogenase activity and by a monoclonal antibody. Macrophages were scattered in the interstitial tissue closely attached to and mixed with the Leydig cells. They were never found in the seminiferous tubules. The macrophages comprised about 25% of all the cells in the interstitium. Double staining with a vital dye and a marker antibody showed that all the phagocytosing cells were macrophages and that the Leydig cells did not take up vital dyes. Double staining for the demonstration of the 3-hydroxysteroid dehydrogenase activity and the macrophage antigen likewise revealed two distinctly different cell populations. Crude Leydig cell preparations obtained by collagenase treatment of the testis contained macrophages (12–14%). Macrophages were present throughout the postnatal prepuberal development of the testis. Their density was increased in the cryptorchid and irradiated testis.     

Rat Macrophage C-Type Lectin Is an Activating Receptor Expressed by Phagocytic Cells

Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.     

Expression of markers shared between human natural killer cells and neuroblastoma lines

Summary Neuroblastoma is a tumor of neuroectodermal origin arising most commonly from the adrenal medulla. We have examined the ability of several monoclonal antibodies which recognize markers predominantly expressed on human natural killer (NK) cells to react with neuroblastoma cell lines in vivo derived sections of tumor. HNK-1 (Leu 7) is a monoclonal IgM antibody which recognizes a carbohydrate epitope on NK cells and a wide range of tumor cell types. We have shown that HNK-1 recognizes the human neuroblastoma lines SMS-KCNR, SMS-KAN, NMB/N7, and IMR/5. Expression of this antigen on cell lines can be slightly increased by retinoic acid-induced differentiation of the cells. N901 (NKH1), a monoclonal antibody raised against interleukin 2-dependent human NK cell lines also recognizes all human neuroblastoma cell lines examined. This expression is independent of differentiation induction and levels remain unaltered following retinoic acid treatment of the cell lines. Lastly, with monoclonal antibody 49H.8, it has been found that reactivity of the lines is weak until induction of differentiation, after which highly significant increases of reactivity are seen. 49H.8 recognizes several cryptic carbohydrate antigens with varying affinities, shown to identify mouse and rat NK cells. In contrast to other NK markers, human neuroblastoma cell lines did not express significant reactivity with B73.1, Leu 11b, or Leu 18. Immunohistochemical staining of sections of human neuroblastoma tumors correlated with the in vitro findings; however, staining with N901 and 49H.8 was only seen on frozen sections, not paraffin-embedded. The significance of shared NK cell-neuroblastoma/neuron antigens is currently under investigation.